In recent years biosurfactants have attracted increasing interest since they show a number of advantages over surfactants of petrochemical origin in terms of ecological acceptance, low toxicity and sustainability. Biosurfactants also have a huge application potential, for example in pharmaceutical and chemical industry or as emulsifier in cosmetics and foods.
Among the best established biosurfactants are the rhamnolipids, which have been first described more than sixty years ago (Jarvis, F. G., & Johnson, M. J., J. Am. Chem. Society (1949) 71, 4124-4126). Rhamnolipids are glycosides with one (mono-rhamnolipid) or two rhamnose-units (di-rhamnolipid) as the glycon portion and one to three β-hydroxy-fatty acid moieties as the aglycon portion. The rhamnose-moiety and the lipid moiety are linked via an O-glycosidic bond. If a plurality of β-hydroxy-fatty acid moieties is present, they are linked to each other by an ester bond that involves the β-hydroxy group(s). The terminal carboxyl group may be a free carboxylic acid group or a methyl ester. Rhamnolipids are produced by two rhamnosyltransferases encoded by rhlA, rhlB and rhlC. The rhlA and rhlB genes form an operon, encoding subunits A and B of rhamnosyltransferase 1, while rhlC encodes rhamnosyltransferase 2. RhlC is part of an operon together with a gene (PA1131) of so far unknown function. Rhamnosyltransferase 1A is responsible for the synthesis of the fatty acid dimer moiety of rhamnolipids and free 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors for rhamnolipid production. Mono-rhamnolipids are then synthesized by Rhamnosyltransferase 1B, which links a rhamnose molecule to a hydroxyalkanoic acid. Rhamnosyltransferase 2 generates di-rhamnolipis by adding a second rhamnose molecule to mono-rhamnolipids. Rhamnolipids have been found to be produced by a number of different bacteria (for an overview see Abdel-Mawgoud, A. M., et al., Appl. Microbiol. Biotechnol (2010) 86, 1323-1336) and potential Rhamnosyltransferase 1 and 2 genes keep being reported.
Industrial efforts to use microorganisms instead of traditional chemical processes increase, as biocatalysis typically allows the use of more moderate temperatures and ambient pressure and enzymes generally have a high selectivity directing reactions to the formation of a particular product. In fermentation whole cells carry out complex reaction cascades leading to a desired product. A fermentation based process thus only requires downstream processing, whereas upstream operations such as precursor synthesis are mostly dispensable. Nevertheless, bacteria usually do not feature the needed resistance against substrates or even products and capacity to produce precursors. Good knowledge of the metabolic network is thus essential, as metabolic engineering is often the only way of achieving the optimal strain. Unwished generation of by-products can thus be eliminated and substrate utilization can be optimized. Essential when applying metabolic engineering is an adequate organism with a well-known metabolism.
Efforts to produce rhamnolipids in a fermentation process have previously been reported (Trummler et al., Eur J Lipid Sci Technol (2003) 105, 536-571; Cha et al., Bioresour Technol (2008) 99, 7, 2192-2199; Ochsner et al., Appl. Environ. Microbiol. (1995) 61, 3503-3506; Müller et al., Applied Microbiological Biotechnology (2010) 87, 1, 167-174); Cabrera-Valladares et al., Applied Microbiological Biotechnology (2006) 73, 187-194 and Wang et al., Biotechnol Bioeng. (2007) 98, 4:842-53). These approaches have largely been aimed at the use of a non-pathogenic organism, and based on employing Pseudomonas aeruginosa or recombinant Pseudomonas putida, albeit production in genetically modified Pseudomonas is so far marginal. Further, yields have so far been unsatisfactorily and no attempts have been made in achieving control over the variety of rhamnolipids formed. For example, Ochsner et al., overexpressed rhlAB genes and obtained 0.6 g/l in Pseudomona putida KT2442 and suggested this strain to be particularly useful, since it accumulates 3-hydroxy fatty acids which may serve as precursors for poly(3-hydroxyalkanoates) and rhamnolipid synthesis. Furthermore, these authors suggest optimizing rhamnolipid synthesis by medium induction and bioprocess optimization in order to industrially apply such optimized strains. In sum, the carbon yield in Cmol rhamnolipid in relation to Cmol substrate, i.e., Cmol rhamnolipid/Cmol substrate achieved by Müller et al. when using P. aeruginosa PAO1 and sunflower oil as substrate is 0.07, by Trummler et al. using Pseudomonas sp. DSM2874 and oleic acid as substrate is 0.18, by Cha et al. using P. putida KCTC1067 and soybean oil as substrate is 0.17, by Ochsner et al. using P. aeruginosa PG201 and gylcerol as substrate is 0.17 or 0.09 when using P. putida KT2442 and glucose a substrate, by Wang et al. using E. coli TnERAB and glucose as a substrate is 0.07 and 0.06 when using P. aeruginosa PEER02 and glucose as a substrate.
The following table shows relevant carbon yield values (Cmol rhamnolipid/Cmol substrate and % of the theoretical maximum Cmol rhamnolipid/Cmol substrate). Note that when “oils” such as sunflower oil was used as carbon source, the theoretical maximum yield is given in relation to octanoate.
Carbon Yield1% of[Cmolrhamnolipid/theoreticalOrganismCmolsubstrate]maximumReferenceWildtypesP. aeruginosa PAO10.077.6%Müller et al. 2010bPseudomonas sp. DSM 28740.1819.8%Trummler et al. 2003RecombinantsP. putida KCTC 10670.1717.9%Cha et al. 2008P. aeruginosa PEER020.044.4%Wang et al. 2007E. coli HB1010.011.1%Cabrera-Valladares et al. 2006aP. aeruginosa PG2010.1723.9%Ochsner et al. 1995E. coli TnERAB0.079.3%Wang et al. 2007P. aeruginosa PEER020.068.3%Wang et al. 2007P. putida KT24420.0912.7%Ochsner et al. 1995E. coli W31100.045.1%Cabrera-Valladares et al. 2006aE. coli TnERAB0.034.0%Wang et al. 2007P. fluorescens ATCC 154530.022.7%Ochsner et al. 1995E. coli DH5α<0.010.7%Ochsner et al. 1995P. oleovorans GPo10.000.0%Ochsner et al. 1995
However, none of these authors, apart from suggesting optimization of, for example, growth conditions or the carbon source; suggest other ways to optimize rhamnolipid production in a bacterial host. Indeed, in particular Ochsner et al., despite the use of the strong tac promoter which drives expression of the rhlAB operon did not achieve satisfying yields of rhamnolipids and suggested therefore either the optimization of growth conditions or the use of Pseudomona strains which produce large amounts of rhamnolipid precursors.
It is thus an object of the present invention to provide a method of producing rhamnolipids and an organism suitable for such a method that when used in rhamnolipid production overcomes at least one of the draw backs of the prior art. This object is solved by the method and the bacterial host cell according to the independent claims.